Since joining the Surgery Branch, my laboratory has successfully developed a novel high throughput methodology to isolate and expand low frequency, high avidity antigen specific CD8+ T cells from peripheral blood. This technique utilizes a highly sensitive PCR based screening assay that can detect the reactivity of rare T cells in a bulk population. Utilizing this approach, after approximately one week of culture, we have been able to prospectively detect T cells from the natural peripheral blood repertoire of melanoma patients that recognize the gp100154-162 epitope from the gp100 antigen, a process that would have been exceedingly difficult and time consuming with conventional repetitive in vitro stimulation techniques. These cells were then isolated and characterized to possess high avidity for the gp100154-162 epitope, an early effector memory phenotype with high surface expression of CD27, and were generated in the setting of limited antigen and IL-2 exposure. We have translated these findings to a current phase II clinical trial transferring gp100154-162 reactive lymphocytes to patients with metastatic melanoma. Early observations from this trial have been the engraftment of the transferred cells in the host and the development of autoimmune effects, which have not been previously described for this cell population In parallel studies, we have applied this platform to the robust isolation of CD8+ T cells that recognize the cancer-testes antigen, NY-ESO, which is expressed in melanoma, breast cancer, prostate cancer, and to varying degrees in bladder, lung, hepatocellular, ovarian, and thyroid cancers. Future plans include the clinical transfer of lymphocytes that recognize the NY-ESO157-165 epitope, which would extend our current studies of adoptive immunotherapy to a variety of common malignancies.